Intravitreal injection of connexin43 mimetic peptides for the treatment of optic neuropathy using an in vivo retinal ischaemia-reperfusion rat model

Abstract

Optic neuropathy is associated with retinal ganglion cell (RGC) loss leading to optic nerve damage and visual impairment. Uncontrolled connexin43 (Cx43) hemichannel opening plays a role in RGC loss. Thus, inhibition via Cx43 specific mimetic peptides (MP) may prevent further cell death. However, the highly hydrophilic character and poor stability of native peptides can limit efficient delivery in a clinical setting. The purpose of the present study was to improve the stability and permeability of Cx43 MP by chemically conjugation to C12-lipoamino acid (Laa) groups or a sugar moiety as well as encapsulating the peptide within poly(D,L-lactide-co-glycolide) (PLGA) micro- (Mps) and nanoparticles (Nps). In addition, in vivo studies investigated the ability of intravitreal injection of these different formulations to promote RGC survival in a retinal ischaemia-reperfusion rat model. Cx43 MP and its modifications were successfully synthesised with high purity (>95%) and acceptable recovery (23-35%). Conjugation to C12-Laa or sugar did not affect the functionality of Cx43 MP. In addition, stability studies in bovine vitreous showed improved stability of modified peptides up to almost three-fold compared to the unmodified Cx43 MP. The Mps and Nps size was around 9 μm and 113 nm, respectively, and Cx43 MP entrapment efficiency was 97% (Mps) and 70% (Nps). A triphasic release profile was observed with an initial burst of surface-bound Cx43 MP followed by slow release due to polymer erosion and further drug release after complete particle breakdown, with 100% release achieved after 112 (Mps) and 63 (Nps) days, respectively. Mps and Nps displayed a delayed effect on Cx43 regulation and RGC preservation but were unable to down regulate the initial inflammatory response. A combination of both immediate and sustained delivery of Cx43 MP may eventually be optimal to promote RGC survival in chronic disease conditions. The modified C12-C12-Cx43 MP, however, with its increased lipophilicity and vitreous stability, showed the most promising results in preventing vessel leak and inflammation, sparing 80% of RGC. This effect lasted for a minimum of four weeks, suggesting that these molecules may be a clinically relevant neuroprotective tool in the treatment of optic neuropathies.